Field of Invention
This invention relates to novel oil and its components produced by Aureobasidium pullulans and compositions containing these novel oils and its components. These components are liamocins. The liamocin-containing oil and the individual liamocins possess anti-bacterial activity. The liamocins and compositions containing liamocins are used as a topical disinfectant, a transdermal anti-bacterial agent, and a systemically administered antibacterial agent, to name a few. Thus, methods of using the liamocins and compositions containing liamocins to kill bacteria are included in this invention. This invention also relates to methods to produce liamocins with specific head groups.
Discussion of the Prior Art
Aureobasidium pullulans is a polymorphic fungus considered to be a filamentous ascomycete in class Dothideomycetes, subclass Dothideomycetidae (Schoch, et al., Mycologia 98:1042-1053 (2007); Hibbett, et al., Mycol. Res. 111:509-547 (2007)). A. pullulans is well-known as the source of the exopolysaccharide pullulan (Leathers, Pullulan. In: Vandamme, et al., (eds.) Biopolymers, Vol. 6, Polysaccharides II: polysaccharides from eukaryotes, Wiley-VCH, Weinheim, pp. 1-35 (2002); Singh, et al., Carbohydr. Polym. 73:515-531 (2008)). Strains of A. pullulans produce numerous other useful bioproducts, including industrial enzymes such as xylanase (Leathers, J. Ind. Microbiol. 4:341-348 (1989)). Nagata, et al. (Biosci. Biotechnol. Biochem. 57:638-642 (1993)) identified Aureobasidium spp. strains that produced poly(β-L-malic acid) (PMA) from glucose. They observed that these cultures also produced extracellular heavier-than-water “oils” (Nagata, et al. (1993)). A partial structure suggested the oils were 3,5-dihydroxydecanoyl and 5-hydroxy-2-decenoyl esters of arabitol and mannitol (Kurosawa, et al., Biosci. Biotech. Biochem. 58:2057-2060 (1994)). Oils from these strains were also observed to exert an antiproliferative effect on cancer cell lines (Isoda and Nakahara, J. Ferment. Bioengin. 84:403-406 (1997)).
An analysis of the oil revealed a set of novel compounds named liamocins (Price, et al. Carbohyd. Res. 370:24-32 (2013)). Four types of liamocins (A1, A2, B1, and B2) were previously identified by Price, et al. (2013). The previously disclosed liamocin A1 and B1 have a single D-mannitol headgroup attached to three or four 3,5-dihydroxydecanoic esters, respectively. The previously disclosed liamocin A2 and B2 are similar in that they have a single mannitol headgroup attached to three or four 3,5-dihydroxydecanoic esters, respectively, but the first dihydroxydecanoic ester, which is attached directly to the mannitol headgroup is 3′-O-acetylated. See FIGS. 1A, 1B, 1C, and 1D for the structures of mannitol-liamocin A1, mannitol-liamocin A2, mannitol-liamocin B1, and mannitol-liamocin B2.
Exophilin A is structurally similar to liamocin. Exophilin A is produced by the marine microorganism Exophiala pisciphila and has antimicrobial activity against certain Gram-positive bacteria (Doshida, et al., J. of Antibiotics 49(11):1105-1109 (1996)). Doshida, et al. (1996) reported that exophilin A was most active against Enterococcus faecium and E. faecalis (MICs of 12.5 and 25 μg/ml, respectively) and against three strains of Staphylococcus aureus (MIC of 50 μg/ml), but did not inhibit growth of Streptococcus epidermis (MIC >100 μg/ml). Of note, E. pisciphila strain NBRC 108784, subject of Doshida, et al. (1996) has been reclassified as A. pullulans by the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) in Japan.
Streptococcus is a genus of ubiquitous Gram-positive bacteria with both pathogenic and nonpathogenic species. Some species are commensal members of the normal flora of the skin, intestine, and respiratory tracts of animals and humans. Others are recognized as etiologic agents of a number of diseases in veterinary and human medicine. S. agalactiae is the most common and recognized species of streptococci that causes mastitis in dairy cattle (Keefe, Can. Vet. J. 38(7):429-37 (1997)) and disease in certain fish and other agriculturally important animals. Although less common, S. uberis can also cause mastitis problems, spreading to cows from the environment or between cows during milking (Leigh, The Veterinary Journal 157(3):225-238 (1999)). S. suis is an emerging zoonotic pathogen associated with diseases like septicemia, pneumonia, and endocarditis in pigs (Lun, et al., The Lancet Infectious Diseases 7(3):201-209 (2007)). In human medicine, infections by species of Streptococcus cause numerous diseases including, but not limited to, pharyngitis (strep throat), impetigo, sepsis, toxic shock, and necrotizing fasciitis (Cleary, et al., Medically important beta-hemolytic streptococci. 3rd ed. in: The Prokaryotes, (Ed.) M. Dworkin, Vol. 4, Springer. New York, pp. 108-148 (2006)).
Mastitis refers to inflammation of the mammary gland. Physical, chemical and usually bacteriological changes in the milk and pathological changes in the glandular tissue characterize it. These glandular changes often result in a number of symptomatic conditions such as, discoloration of the milk, the presence of clots, and the presence of large numbers of leukocytes. Clinically, mastitis is seen as swelling, heat, pain and induration in the mammary gland often resulting in deformation of the udder. In many cases the diagnosis of subclinical infections has come to depend largely on indirect tests which depend on the leukocyte content of the milk or somatic cell count (SCC). Mastitis can occur when the animal's teat and/or udder has been infected by any of several species of bacteria. Streptococcus spp., in general, and S. agalactiae, S. uberis, S. bovis, and S. dysgalactiae, in particular, are a major cause of mastitis. Enterococcus faecalis can also cause mastitis.
In a dairy herd, 50% of the intra-mammary infections develop during the nonlactating period of the lactation cycle known as the dry period. The standard management practice to reduce the number of infections during the non-lactating period is to administer systemically a long-lasting and concentrated antibiotic preparation immediately after the last milking preceding the dry period. While this procedure has been effective, it is undesirable from a food safety standpoint. Because of human error, milk tainted with the antibiotic occasionally becomes commingled with milk intended for the market; and, subsequently, the milk must be discarded causing significant economic loss to the dairy producer. In addition, the widespread use of systemic antibiotics is undesirable from the standpoint of creating a population of microorganisms in cattle which may be resistant to antibiotics typically used to treat cattle disease.
Dairy cows are milked for about 305 days and go into a period of non-lactation (dry period) for about 60 days. During the first several days of the dry-period, the mammary gland is very susceptible to infection because the white blood cell count in milk is very low (McDonald and Anderson, Am. J. Vet. Res. 42:1366-1368 (1980)). If the gland should become infected, the heifer lacks a sufficiently active immune system within the udder, teats, and mammary glands to keep the bacteria from growing and ultimately resulting in an intramammary infection. It takes about 4-6 days from dry-off for the white blood cells in milk to reach levels that are protective.
In addition to mastitis, Streptococcus spp. are involved in many diseases in many different animals. For example, while rumen acidosis may not be initially caused by S. bovis, explosive S. bovis growth within the rumen occurs during rumen acidosis, resulting in the rumen's pH dropping even further. Several species of Streptococcus are known pathogens for marine and freshwater fish, including, but not limited to, S. difficilis, S. milleri, S. parauberis, S. agalactiae, and S. iniae. S. pneumoniae causes pneumonia in animals and humans. S. agalactiae causes septicemia in humans and farm animals. S. pyogenes causes strep throat, necrotizing fasciitis, and other diseases in humans, while S. mutans causes dental caries.
There is a need for a new method of preventing or treating infections by Streptococcus spp., in general, and specific species of Streptococcus. There is also a need for chemicals and compositions that can kill Streptococcus spp., in general, and specific species of Streptococcus, when applied to a surface containing the bacteria.